ABSTRACT Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are generally incurable hematologic malignancies, originating from aberrant hematopoietic stem cells (HSCs). These diseases occur mainly in the elderly and are preceded by an often-unrecognized precancerous phase, which can last for years and is hallmarked by a progressively changing HSC compartment. Such molecularly and functionally diverging stem cells, termed pre-leukemic stem cells (pre-LSCs), harbor a higher propensity to undergo malignant transformation. Mechanisms that separate the healthy aging process from cancer evolution are incompletely resolved. This gap in our knowledge poses a significant challenge for the development of curative therapies for myeloid malignancies for which cure rates have mostly remained below 30% over the last decades. Our study will investigate the role of chaperone-mediated autophagy (CMA), a highly selective subtype of autophagy, which has not been systematically characterized in the hematopoietic system thus far. Contrary to macroautophagy, CMA specifically degrades proteins containing defined recognition motifs (?KFERQ?, and variants), and maintains integrity and proper function of many cell types during chronic stress and aging. CMA declines in and critically contributes to several age-associated pathologies, yet its role in leukemogenesis and cancer stem cell evolution is unknown. Using new genetic mouse models, we have obtained exciting preliminary data demonstrating that CMA sustains the function of healthy HSCs. Furthermore, we found that impairment of CMA increased pre-LSC function that appears to be linked with increased levels of reactive oxygen species, accumulating in CMA-deficient stem cells. For this study, we hypothesize that CMA protects against stem cell dysfunction and malignant transformation. We will utilize a complementary model set consisting of genetic mouse models, human cell lines and primary patient-derived cells for the study of (1) CMA activation patterns in healthy HSCs and pre-LSCs (leveraging a CMA reporter mouse and lentiviral CMA biosensors for primary human cells), (2) molecular and (3) functional consequences of CMA inactivation and stimulation. We will also test a new chemical CMA activator tool compound for its efficacy to promote healthy HSCs and impair pre-LSCs, which could be further developed for clinical use. Our study will provide new insights into a molecular mechanism declining during aging and predisposing adult tissue-specific stem cells to malignant transformation, which will have important implications for the development of new therapeutic strategies for targeting autophagy in myeloid malignancies, and possibly other stem cell-derived cancers.